Project Summary and Abstract The small regulatory RNA pathway centered on Piwi (P-element-induced wimpy testis) proteins and their bound Piwi-interacting RNAs (piRNAs) silence transposons and other targets to maintain genome integrity in animal germlines. Disruption of the piRNA pathway causes elevation of transposon levels and defects in germline development, eventually resulting in sterility of the animals. The piRNA pathway thus functions as guardians of the germline genome to ensure genetic information is passed onto the next generation. Although the crucial function of piRNAs in germline development is evident, the factors and mechanisms mediating piRNA biogenesis remain elusive. Biogenesis of the piRNAs starts by transcription of long single-stranded RNAs from defined genomic regions termed piRNA clusters. Precursors transcripts are likely processed into shorter primary precursors. However, profiles and mechanisms of the processing for primary piRNA precursors are not well elucidated. The primary precursors are further processed by the mitochondria-associated enzyme Zucchini to generate pre-piRNAs, which are loaded onto Piwi proteins where they undergo 3?-end processing and methylation to become mature piRNAs. Toward our research goal to clarify the biogenesis pathway of piRNAs, we have been utilizing Bombyx mori ovary-derived BmN4 cells which endogenously express Piwi proteins and piRNAs. The scarcity of piRNA-expressing culturable cells make BmN4 cells suitable and well used for piRNA biogenesis research. In the cells, piRNAs are produced abundantly from tRNAs, and we recently revealed the biogenesis mechanism of the tRNA-derived piRNAs, in which 5?-tRNA halves, not mature tRNAs, become direct precursors for piRNAs. Our finding of a 2?,3?-cyclic phosphate (cP) at the 3?-end of 5?-tRNA halves prompted us to hypothesize that cP-containing RNAs (cP-RNAs) form piRNA precursors. Our hypothesis was strengthened by our further analyses showing that, in BmN4 cells, cP-RNAs are abundantly accumulated at the outer membrane of mitochondria where piRNA biogenesis takes place, and that these cP-RNA sequences are extensively overlapped with piRNA sequences. cP-RNAs form a hidden layer of the transcriptome because standard RNA-seq is unable to capture them. We have developed a cP-RNA-seq method that can selectively sequence cP-RNAs, and we will utilize it to comprehensively identify the cP-RNAs expressed in Bombyx BmN4 cells, Drosophila Kc167 cells, and mouse testes in order to investigate their expressional relationship with piRNAs (Aim 1). We will further elucidate the interaction of cP-RNAs with Piwi proteins and a piRNA biogenesis factor BmPapi (Aim 2), and characterize an RNase producing cP-RNAs in piRNA biogenesis (Aim 3). The proposed studies will fuel our novel research efforts to understand the mechanism of piRNA biogenesis by focusing on currently-uncharacterized cP-RNAs, and support biomedical goals to understand the pathogenesis of reproductive system diseases and cancers that ectopically express Piwi proteins.